Project 1 Project Leader: Van Vactor, David L. Abstract It has become increasingly clear that neuronal connectivity, function and plasticity all rely on the post- transcriptional regulation of gene networks. Among newly discovered classes of regulatory factor likely to be vital for regulating mRNA stability and translation in neurons, small non-coding microRNAs (miRs) have tremendous potential to shape the gene expression landscape. This large class of sequence-specific regulatory molecules is highly expressed in the nervous system. However, very few miR functions have been examined in vivo where the relationship between neural circuit architecture and behavioral outputs are maintained. To address this challenge, we have recently created tools to manipulate miR function with spatial and temporal specificity, in a model organism ideal for the study of neural circuits that control a wide variety of behaviors. Drosophila offers an increasingly sophisticated set of genetic approaches and reagents that are complementary and enabling to the new tools we have produced. As part of a carefully orchestrated set of collaborations using multiple robust and fundamental behaviors in this organism, we set out to map the miRs essential in the neural circuits controlling locomotion, sleep and associative memory. Using the larval NMJ as a model synapse for genetic validation ideal for miRs that may act in multiple neural circuits or multiple stages of postembryonic development, we have shown that null mutations recapitulate 85% of our miR-SP bouton phenotypes tested to date. Our functional screens reveal four conserved miRs likely to shape the development and plasticity of neural circuits: the miR-13 and miR-92 families, miR-190 and let-7. With an array of techniques, many of which we are developing with and/or learning from our collaborators, we will determine which of these miRs are essential for neurotransmission and activity-induced remodeling at the synapse. We will use new genetic tools to define the spatial and temporal logic for each miR function. We will then use a state-of-the-art combination of transcriptome sequencing and computational informatics, followed by use of in vivo activity sensors and functional validation, to discover the downstream mechanisms for each miR that intersects our coordinated screens. With an initial focus on the miR-92 family, we have obtained preliminary informatics and expression data identifying several highly conserved target gene candidates that have been implicated in developmental and activity-dependent synaptic signaling pathways. Once we dissect the cellular and molecular mechanisms for each gene, we will then compare each miR-target relationship across our model circuits to determine which mechanisms are context-specific, and which are widely used across the brain. This combination of comprehensive discovery science and comparative molecular anatomy will address fundamental and timely questions in a way that is not yet possible to achieve in mammalian models, but is likely to identify regulatory strategies highly relevant to many neural circuits.